Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Terminal deoxyribonucleotidyl transferase activity in acute undifferentiated leukemia.

Guanosine, rather than its methylated derivative, was found to be the inverted nucleoside present in the 5′ terminal capping structure of insect oocyte messenger RNA. Since methylation of the terminal guanosine at the 7 position is necessary for the initiation of protein synthesis in eukaryotes, this evidence suggests that the translational inactivity of the mRNA prior to fertilization may be associated with the absence of methylation.     

Dependence of the maximum temperature for growth of Saccharomyces cerevisiae on nutrient concentration

Anacystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30°C. Such cultures ahd a normal pigment composition and alkaline phosphatase was detectable at low specific activities only.The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 M (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate.Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess.Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.Abbreviations Used pNP pnitrophenol - pNPP pnitrophenylphosphate     

Light and Electron Microscope Study of Dunaliella primolecta Butcher (Volvocida)*

SYNOPSIS. Light and electron microscope observations on Dunaliella primolecta Butcher from logarithmic and stationary phases of batch cultures are correlated. Except for the lack of a cell wall the fine structure has typical volvocid features. The transition from logarithmic to stationary phase is marked by changes in content and size of cytoplasmic vacuoles, accumulation of cytoplasmic lipid, accumulation of starch in the plastid matrix, and by the formation of autophagosome-like bodies. The organelles in stationary-phase flagellates are closely packed together because of the cytoplasmic lipid and starch-distended chloroplast. Organisms from logarithmic phase have an abundant ribosome-packed groundplasm supporting the organelles. In the cytochemical demonstration of acid phosphatase activity, Golgi cisternae and smooth and coated Golgi vesicles contain Gomori reaction product. The possible roles of the Golgi apparatus in this flagellate are discussed.     

Recherches Cytochimiques sur la Microsporidie Nosema bomycis au cours de son Développement chez le Ver à Soie (Bombyx mori)*

RESUME. La Microsporidie Nosema bombycis, Protozoaire parasite agent de la pébrine du ver à soie, a étéétudiée cytochimiquement à la fois en microscopie photonique et électronique. Les examens ont porté sur la détection et la localisation des acides nucléiques (ADN et ARN), des polysaccharides, de la phosphatase acide, au cours des différents stades du développement dans les cellules de I'hôte (du schizonte à la spore). Les principaux résultats concernent les observations relatives aux polysaccharides et à la phosphatase qui ne sont détectés qu'au stade de la spore et ne sont pas observés au stade du schizonte. Les polysaccharides sont présents au niveau du sac polaire, du filament polaire et sur la membrane cytoplasmique; la phosphatase acide est localisée au niveau du sac polaire, du filament polaire et dans la vacuole postérieure. SYNOPSIS. Nosema bombycis, agent of pebrine disease of silkworm, was studied cytochemically, using both light and electron microscopy. Presence of nucleic acids (DNA and RNA), polysaccharides, and acid phosphatases was demonstrated and localization of these substances was determined in various stages of the parasite (from the schizont to the spore). DNA and RNA were detected in all these stages. Polysaccharides and acid phosphatase were found in the spore but not in the schizogonic stages. Polysaccharides were detected in the polar cap, the polar filament, and the limiting membrane of the cytoplasm of the spore. Acid phosphatase was found in the polar cap, the polar filament, and the posterior vacuole.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异     

Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS‐induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras‐induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation‐induced inactivation. Along those lines, Prdx1 tumour suppression of Ras‐ or ErbB‐2‐induced transformation was mediated mainly via PTEN.